Allosteric modulation of GPCR-induced β-arrestin trafficking and signaling by a synthetic intrabody

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of β-arrestin (βarr) recruitment and trafficking. For several GPCRs such as the vasopressin receptor subtype 2 (V2R), agonist-stimulation first drives the translocation of βarrs to the plasma membrane, followed by endosomal trafficking, which is generally considered to be orchestrated by multiple phosphorylation sites. We have previously shown that mutation of a single phosphorylation site in the V2R (i.e., V2RT360A) results in near-complete loss of βarr translocation to endosomes despite robust recruitment to the plasma membrane, and compromised ERK1/2 activation. Here, we discover that a synthetic intrabody (Ib30), which selectively recognizes activated βarr1, efficiently rescues the endosomal trafficking of βarr1 and ERK1/2 activation for V2RT360A. Molecular dynamics simulations reveal that Ib30 enriches active-like βarr1 conformation with respect to the inter-domain rotation, and cellular assays demonstrate that it also enhances βarr1-β2-adaptin interaction. Our data provide an experimental framework to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can be potentially integrated in the paradigm of GPCR-targeted drug discovery.

The ideas are very easy to follow as they are clearly explained step-by-step and the flow follows a logical sequence. Additionally, sufficient context have been provided to understand the rationale of each experiment.

References:
The references are appropriate.
Reviewer #2 (Remarks to the Author): Review of "Allosteric modulation of GPCR-induced β-arrestin trafficking and signaling by a synthetic intrabody" by Baidya et al.
In this manuscript the authors present a detailed investigation of the how contact with binding partners controls the conformation and function of β-arrestin 1. First the authors evaluate how the binding of phosphorylated peptides either with or without a key phosphorylated residue (at position T360) or the synthetic intrabody (Ib30) impact arrestin conformation, as measured using a limited trypsin proteolysis assay. These assays suggested subtle conformational differences were induced through arrestin binding as demonstrated by differences in the extent and site of trypsin proteolysis. These differences were evaluated in the context of previously published structural data as well as new molecular dynamics studies. The authors next evaluated functional consequences of the interaction between the synthetic intrabody and beta-arrestin in live cells. Here they found that Ib30 expression facilitates endosomal trafficking upon V2R T360A activation, a very surprising and interesting finding. This effect of Ib30 was shown to correlate with an enhanced interaction between beta-arrestin and beta-adaptin, which is known to facilitate internalization.
Overall the paper is well written, the experiments are carefully performed and interpreted appropriately, and the biological implications of the findings are high. Some of the arguments made regarding structural changes are a bit speculative. The importance of understanding (and controlling) receptor internalization mechanisms is extremely high. As such, I think the manuscript could be suitable for publication provided some major and minor concerns are addressed.
Major comments: *Receptor internalization (and signaling from internalized compartments) is increasingly being recognized as an important element in determining the biological output of receptor activation. The system described here seems to offer a way to toggle internalization on/off. However, this manuscript doesn't address whether the internalization observed for bArrestin in cells expressing V2R-T360A and Ib30 also correlates with receptor internalization. The authors describe already use a whole cell ELISA assay for looking at receptor levels. This assay (or something related) should be used to see whether Ib30 also induces receptor internalization for V2R-T360A. This would substantially improve the impact of this manuscript. *In Figure 3 the authors use luciferase complementation to measure beta-arrestin recruitment to the cell surface. In Figure 4 the authors use BRET to measure arrestin recruitment to the endosome. It would be better if the authors could run the same kind of assay for both plasma membrane and endosomal recruitment. Plasma membrane anchored GFP plasmids are available for BRET. *In figure 5b the frequency of different angles for interdomain rotation is shown as a result of molecular dynamics simulation for beta arrestin in complex with various partners. Predicting conformations for dynamic proteins is tricky and predicting binding for antibody (fragments) is also tricky. Can the authors provide more evidence that the modeling they performed for bArr1/VwRppT360A/Fab30 is likely to be reflective of reality? As it stands now, I feel the manuscript makes too strong of a conclusion from a modelling experiment that is filled with uncertainty.
Minor comments: *For all statistical comparisons a one-way ANOVA is used to provide p-values but this test requires posthoc analysis (like Tukeys) to inform you which groups are actually statistically different from each other. Could the authors specify which post-hoc tests were used? It would also be helpful to specify which two groups are being compared for each p value (I think I can follow but I don't want to have to assume) *Why is V2RppT360-2¬ called this? It doesn't have a Thr at position 360? *For limited proteolysis experiments can the authors demonstrate that the phosphopeptides are not being degraded at different rates? *In Figure 1c why is there a band labeled Gly (-8) to Arg418 that doesn't appear in Figure 2c? *What accounts for the reduced ability of ScFv30 to immunoprecipitate beta-arrestin 1/V2RppT360 complex ( Figure 2a)? Is the phosphopeptide used at a saturating concentration for beta-arrestin here (what is the KD for this interaction?) Is it because ScFv30 has a lower affinity for beta-arrestin 1/V2RppT30 complex? If this is the case could you explain it explicitly in the text *In Figure  *It seems the level of Ib-CTL/30 is variable in Figure 4c and no conventional loading control is shown. Can the authors comment on this?
*Could the authors comment on whether endogenously expressed beta-arrestins play any role in the observed results for cell-based assays? *Why is the non-specific band observed in Figure 6A not seen in other blots?
Reviewer #3 (Remarks to the Author): In their manuscript, the authors investigate the effect of the intrabody30 (Ib30)on the activity state of arrestin bArr1 bound to the V2R. The manuscript builds on previous observations published in Nat. com., that the T360A mutation at the C-terminus of the receptor leads to a loss of arrestin translocation to the endosomal membrane due to a prominent shift in arrestin from active to inactive. In the current manuscript, they investigate the effect of Ib30 binding to bArr1 to reverse this effect. For this purpose, they use various biochemical and biophysical in vitro and in silico methods. In their experimental setup the authors find that Ib30 rescues agonist induced ERK 1/2 MAP kinase activation by the T360A mutation. Moreover, that Ib30 enhances interaction of bArr1 with b2-adaptin. To the reviewer, the experiments appear well presented and convincing.
In the second part of the manuscript, structural explanations are provided. This portion is of particular importance for the argumentation, as it establishes a reference to the structural level. However, in the opinion of the reviewer, this part is not elaborated far enough and should be deepened or better documented in order to make it accessible and understandable to the broad readership of the journal. The MD simulations indeed seem to have the potential to reproduce and substantiate the observations at a structural level. The interdomain rotation angle used here is a recognized measure for classifying a state as active or inactive. In the present manuscript, the authors use this very measure to support the above hypothesis. The results of three simulation repeats are summarized in Fig. 5B in the form of a histogram. The current presentation of the data appears to the reviewer to be inadequate because significance is not apparent from the provided histograms. However, the data provided to the reviewer on request during the review process allows the following assessment of the analyses presented: The MD simulations indeed confirm the hypothesis, since within the limitation of non-sufficient sampling, the WT arrestin tends to be in active conformation, the T to A mutation tends to be in inactive conformation, and the latter mutation in complex with intrabody30 (Ib30) again tends to be in active conformation. From the running avarages now available to the reviewer, it is clear that the arrestin does not remain in one state in each case, but rather fluctuates between active and inactive, which has not previously been described in this way in the results section. This fluctuation proves that the respective tendency to active or inactive, is not an artifact of a single simulation, but rather and despite the limited simulation time describes an actual property of the system. The reviewer suggests to integrate the histograms into the illustration of the running averages, for example the present illustration turned by 90°, at the end of this. This would already make clear from the figure that the undoubtedly different tendencies are due to an overall limited amount of data -in this case three short simulations -and do not represent the actual ensemble. In doing so, the dimensions of the Y-axes should also be aligned for all three systems to ensure direct comparability. Furthermore, a space between the individual trajectories should convey even better to the reader that we are dealing with three independent runs and not a continuous simulation. In any case, this limitation, which most simulation work is subject to due to the limiting and costly calculations, should be sufficiently described in the paper.
The reviewer wonders why the structural interpretations from Figs. 2E,F and 5A based on previously published crystal structures, were not supported with MD simulations. On the one hand, the view on static structures does not allow optimal statistics of the contacts, i.e. it is not clear whether the structural interpretations mentioned in the text, which are partly due to side chain movements of single amino acids, are equally observable in a dynamic setup. Second, and more importantly, the described effects of the T to A mutation on the activation or deactivation of arrestin or the effect of the intrabody30 (Ib30) thus do not experience a sufficient structural explanation. A more detailed study of the structures using MD, or further analysis of the existing MD data, should allow the authors to describe the long-range allosteric effects underlying their observations. Naturally, the MD part would thus assume an even more importance in the manuscript.
Finally, the methodology section appears to the reviewer to be in urgent need of revision, as essential information cannot be found here. It was neither helpful for the review, nor does it facilitate the readability of the manuscript, if essential details are not found in the manuscript but in another publication, which is merely referred to. The method used to measure the interdomain rotation in the trajectories is thus not transparent, mainly because the paper they cite does not explicitly state information such as: which axis they use to measure the rotation angle or which reference points (selected residues/atoms or center of mass/center of mass of the selected residues?). Likewise, the information on the PDB-entries, which provided the structure coordinates for the respective simulations, is missing. What changes were made to the structures (mutations, etc.)? Thus, it was laborious for the reviewer to gather this information from other parts of the manuscript. The reader should be spared this effort. A final question arises from the data in the methodology section. The authors used the NVT ensemble for the production runs, which is rather unusual. Why were this ensemble chosen?
In summary, this manuscript describes work with potential interest for the broad readership of the journal. The experimental part of the work seems convincingly elaborated to the reviewer. The structural level is of great importance for the verification of the biochemical analyses and substantial for the understanding of the effects described here. There are still deficiencies here, but these could be solved within the interdisciplinary orientation of the authors. It is important for the reader to understand not only that the T to A mutation has an effect on the dynamic equilibrium of inactive to active arrestin states, but also how this effect comes about structurally and how it can be reversed by binding of intrabody30 (Ib30). At this point, further analysis seems neccesary. Finally, the MD part should be described and documented in more detail. The working group around Jana Selent has created a useful basis for sharing MD data with the implementation of GPCRMD. I would expect her to use this to share the trajectories analyzed here with the reviewers adequately and subsequently make them available to the community in the spirit of the FAIR principles.
Reviewer #4 (Remarks to the Author): The submitted manuscript by Baidya et al. entitled "Allosteric modulation of GPCR-induced β-arrestin trafficking and signaling by a synthetic intrabody" is a well written manuscript. However, during the entire review process of the manuscript, I had the feeling I have read this story somehow, at least in large part, before.
Major concern: 1) The mutant V2R-T360A and its effect on arrestin recruitment and V2R Trafficking was published before in Ref 10 (by Dwivedi-Agnihotri et al. in 2020 see figure 5 in that paper with arrestin recruitment, trafficking and ERK activation). So what is new here except a bit of ERK stabilization by Ib30 2) What the authors investigate in figure 1 and 2 is the relative conformational change of arrestin by a limited tryptic digest, an established technique since 2006. However, this technique is limited but can show global differences. Since there is no loading control available in these provided gels any conclusion on kinetic effects for the digest will depend on the amount of arrestin added in the individual sample and this is not quantified, at least I did not see this. Hence, this part of discussion is rather weak if not over interpreted.
3) In figure 4 B the V2R wt conditions without and with Ib30 seem to be only shifted in parallel upwards, please take this into consideration in the discussion. The effect might not be as big as described if the two curves would start from the same basal level…. Figure 5 is of concern as well. The modeling is nice but the major effect of Ib30 stabilizing the active conformation of arrestin was shown before using Fab30, the parental antibody of Ib30. This was shown by stabilizing NMR signals of arrestin (cited as Ref. 26). Hence, again the novelty is limited since this result was to be expected, although it is great to see this stabilization in living cells and not only NMR. 5) In figure 6 C the V2R T360A conditions without and with Ib30 seem to be only shifted in parallel upwards, this needs to be taken into account. If the curves would start at the same basal level they would be identical and Ib30 had no effect for the T360A mutant… Minor points:

4)
For this manuscript it would also be helpful to clearly state in the manuscript the differences or similarities between T360A-1 and -2 and when they can be summed up to T360A to follow the authors' throughout the manuscript.
Please make sure that all needed information about statistical analysis is clearly and consistently stated in all figure legends etc. throughout the manuscript.
Line 27, 63/64 vasopressin 2 receptor or vasopressin type 2 receptor Line 76 previously described intrabody (Ib30)-based sensor (hyphen) Line 127 additional information to the CONTACT/ACT program would be nice in order to make it more understandable to readers who are not so familiar Line 211 Please elaborate on the ear-domain of β2-adaptin for the less conversed readers -please indicate the starting and end numbers/positions of the shown amino acids in Fig. 1B -either reduce the shown trypsin digest data in D (because it seems like the data are shown twice for the different trypsin : βarr1 ratios) or make clear why it is important to show both/ point out what is the important difference/message to take from it as a reader -of note: significance level is set to * p < 0.1 instead of 0.05… (which is not the case in Fig.2 for example) Figure 2 -it would be nice to have the probes in 2C consistently with 1C to make it easier for the reader -please indicate whether T360-1 or -2 are shown in Fig.2D or whether it is a representation for both, the Fig.2 focusses on T360-1 in E and F, is this also the case in D? -in figure 2c please also indicate if the big band above 29kDa is the ScFc30 or something else -since the authors only use dark receptor constructs, they should include at least one control in the supplements where they do not co-express the receptor of interest to clearly show that the observed effect is dependent on the presence of the receptor -in Figure 4D, the statistical comparison of V2R-T360A + Ib30 to the WT receptor would be also interesting to add statistical relevance to the statement "it robustly enhanced the level of phosphorylated ERK1/2 upon agonist-stimulation, nearly to that of the V2RWT" (line 177/178) Figure 6 -please include statistical analysis for Fig.6 to support the statement "There was robust interaction between βarr1 and β2-adaptin for the V2RWT upon agonist-stimulation in the presence of control intrabody, while the response was significantly lower for V2R T360A" (line 221-223) and also in lines 224-227. Figure S1 -the quantification of the 48kDa band seems to be missing in Fig. S1 and the information of the quantified time point (B) -of note: significance level is set to * p < 0.1 instead of 0.05… (which is not the case in Fig.S2 for example) Figure S2 -number of independent experiments not indicated Figure S3 -description of graphs inconsistent, kDa information missing Figure S4 -was the significance level set to ns p> 0.1 or > 0.05 here? Figure S5 -no statistical analysis of cAMP response and surface expression quantification, but claim in main text "suggesting that the intrabody does not significantly influence agonist-induced Gαs coupling ( Figure  S5A My overall conclusion it that this paper is written well, but the story is rather confirmatory and does not hold any surprises or unforeseeable results. 3 In this study, the authors characterise the ability of intrabody30 (Ib30) to rescue β-arrestin1 4 translocation to endosomes impaired by a mutation of a phosphorylation site in the carboxy-5 terminal region of the vasopressin type 2 receptor (V2R) previously reported by the same 6 group. The authors demonstrate that Ib30 also rescues agonist-induced ERK1/2 7 phosphorylation by the mutant V2R (V2R T360A ), an event downstream and dependent of β-8 arrestin1 translocation to endosomes. Molecular dynamics simulations previously suggested 9 that V2R T360A reduces the fraction of active β-arrestin1 conformation and in the present study 10 elegant in vitro techniques were used to demonstrate that Ib30 rescues β-arrestin1 11 translocation to endosomes and agonist-induced ERK1/2 phosphorylation by enriching the 12 active-like conformation population of β-arrestin1. The authors also demonstrate that Ib30 13 enhances the interaction between β-arrestin1 and β2-adaptatin, which is a prominent 14 mechanism that drives receptor internalisation. They hypothesise that Ib30-mediated increase 15 of β-arrestin1/β2-adaptatin interaction is the possible mechanistic basis linking active β-16 arrestin1 enrichment and improved β-arrestin1 translocation to endosomes induced by Ib30.

17
Overall these exciting findings provide a proof-of-concept that conformation-specific 18 intrabodies represent a novel molecular tool to allosterically modulate agonist-induced 19 trafficking of β-arrestins and therefore constitute an attractive solution to enhance this pathway 20 in a therapeutic context. 21 We thank the reviewer immensely for her/his time to review our manuscript so thoroughly and 22 for her/his positive comments. 23

Validity: 24 25
The interpretation and conclusions are based on solid data obtained from both in vitro and 26 cell-based assays. The authors frequently use several complementary approaches to 27 measure a given parameter, which strengthens the conclusions and considerably reduces the 28 risk of conclusions based on artefacts. 29 We thank the reviewer for her/his positive comments. 30

Significance: 31 32
Endosomes represent a fantastic signalling platform where specific cellular functions can only 33 occur from these intracellular compartments. For some receptors, ERK1/2 activation is an 34 example, but since the past decade, a growing number of studies report a major functional 35 role of G protein signalling from endosomes in cellular physiology. As Ib30 induces an 36 enrichment of fully-active β-arrestins promoting their trafficking to endosomes, this intrabody 37 could theoretically be used to positively modulate downstream signalling dependent of the 38 presence of β-arrestins in endosomes by any GPCR of interest (not only V2R). Supporting this 39 idea, the authors report that Ib30 efficiently recognises β-arrestin1 in complex with several 40 native GPCRs (ref. 12,22). Some studies report that β-arrestin binding to class B receptors in 41 endosomes potentiates endosomal G protein activation by these receptors. Therefore, by 42 promoting endosomal trafficking of β-arrestin, Ib30 could 43 represent an interesting tool to increase endosomal G protein signalling of these receptors, 44 and consequently selectively potentiate their downstream cellular functions. Additionally, by 45 its ability to recognise the active conformation of β-arrestin, Ib30 could potentially be utilised 46 in the context of drug discovery to screen compounds stabilising a receptor conformation 47 promoting endosomal β-arrestin signalling. Overall, the present study will certainly be of great 48 significance to the field of G protein-coupled receptor (GPCR) signalling from fundamental 49 research to investigate the role of endosomal β-arrestin signalling to applied research to 50 design new therapeutics targeting cellular functions downstream of β-arrestin signalling from 51 endosomes. 52 We thank the reviewer for her/his positive comments and appreciation of our work. 53

Data and methodology: 54
Enough details are provided in the methods for the work to be reproduced. The methodology 55 is overall elegant and adapted to the questions investigated. The authors combine a well-56 balanced ensemble of in vitro techniques, cell-based assays and computational approaches 57 to answer their questions. In several situations, the authors combine different approaches 58 together to answer a given question. The work certainly meet the expected state-of-the-art 59 standards in the field. 60 We thank the reviewer for her/his positive comments and appreciation of our work. 61

Analytic approach: 62
The analytical approach is appropriate. However, in Fig. 1D, as data are normalized to 63 V2Rpp WT , the % band protection to this condition is always 100. Consequently, the variance 64 between the 4 experimental groups is not homogenous and therefore an ANOVA cannot be 65 performed. In that situation, a one-sample t test and comparison to 100 would be the proper 66 statistical test. Same thing for Fig. 2B, 4D, 6B, S1B, S2B, S3A, and S4A. 67 We thank the reviewer for her/his positive comments and appreciation of our work. 68

Suggested improvements: 69
The overall quality of the manuscript is excellent. I do not recommend any additional 70 experiment. However, I have the following minor concerns to improve even more this 71 manuscript: 72 73 -Lines 63-65: While cumulative phosphorylation on GPCRs is believed to determine the 74 affinity of β-arrestin interaction with the receptor, any potential explanation for why mutation 75 of a single phosphorylation site in V2R (T360A) drastically alters β-arrestin trafficking patterns? 76 Are there additional evidence in the literature of such maximal effect with only a single 77 phosphorylation site mutated for other receptors? It would be interesting to elaborate a bit on 78 this aspect as V2R T360A is central to the present manuscript. 79 The dramatic alteration of the βarr trafficking pattern upon Thr 360 Ala mutation likely arises from 80 the disruption of a key salt-bridge with Lys 294 in the lariat loop and resulting conformational 81 change in βarrs. Following the reviewer's advice, we have now included this information in the 82 revised text (page 3-4, line 71-73). We have now also cited an additional reference on the 83 apelin receptor, which also shows a dramatic reduction in βarr recruitment upon mutation of a 84 single phosphorylation site (new reference 12 in the revised manuscript). 85 -In Fig.1B, it would be useful to indicate with an arrow the residue 360 as it takes a few second 86 to figure out where it is. 87 Following reviewer's advice, we have now indicated residue Thr 360 in the revised Figure 1B. 88 -The majority of paragraphs from the results start with "In order to ....". Please use alternative 89 ways to start these paragraphs as it distracts from the interesting story. 90 Following reviewer's advice, we have now revised the text to avoid the repetition of the same 91 starting sentence in different paragraphs. 92 -Lines 140-142: The fact that the authors did not observed any measurable effect of Ib30 on 93 G protein-mediated cAMP responses is a bit unexpected. If Ib30 promotes β-arrestin 94 trafficking to early endosomes, we should expect to see more cAMP produced as megaplex 95 formation increases endosomal Gs activation by a GPCR. Can the authors comment on this 96 and maybe briefly clarify this in this section of the results? 97 This is an interesting point. We are using the GloSensor assay to measure cAMP responses, 98 and this assay saturates rather quickly due to massive amplification of the signal. This may 99 be a plausible reason for why we could not detect a difference in the cAMP response. 100 However, there may be additional possibilities for this interesting observation that should be 101 explored further in future studies. Following reviewer's advice, we have now discussed this in 102 the revised manuscript (page 14, line 326-331). 103 -Lines 176-177: Any idea why Ib30 did not have a potentiator effect on ERK1/2 activated by 104 wild-type V2R if Ib30 increases β-arrestin recruitment to early endosomes? 105 This is also an interesting point. In case of V2R WT , the starting point of the curve is also up-106 shifted and therefore, the net potentiation of the endosomal trafficking is lesser than V2R T360A . 107 We have now clarified this point further by plotting the data differently i.e. differences in BRET 108 signal between the lowest and highest agonist dose ( Figure 6C and 6F). Moreover, as we are 109 measuring ERK1/2 phosphorylation at the 5min time-point, where the response is typically 110 maximal, minor potentiation of ERK1/2 may not be apparent. Following reviewer's advice, we 111 have now discussed this in the revised manuscript (page 13, line 316-322). 112 -Line 244: There is a typo in βarr-β2-adaptatin. 113 This has been corrected. 114 -Line 271-273: It would be indeed interesting to explore in future studies whether the effect of 115 Ib30 observed for V2R T360A is linked to the transition between the partially-and fully-engaged 116 βarr conformations in complex with the receptor. However, before to explore this question, it 117 would be primordial to test if Ib30 rescues other V2R mutants (mutations on phosphorylated 118 Ser/Thr on the carboxy-terminal), such as the mutants characterised in reference 10. 119 This is an interesting point too. In fact, the other V2R mutants characterized in reference 10 120 display either a wild-type-like pattern of βarr trafficking (e.g. V2R S357A and V2R T359A ) or, a 121 complete loss of βarr trafficking (e.g. V2R S362A/S363A/S364A ). Therefore, we focused our attention 122 on V2R T360A in the current manuscript, which displays robust βarr binding but a dramatically 123 altered trafficking profile compared to V2R WT . Following reviewer's advice, we have now 124 mentioned this in the revised manuscript (page 4, line 73-76). 125 -Line 581: Usually one star means p<0.05. The authors wrote *p<0.01. Is this a mistake? 126 Same comment for the figure legend corresponding to Fig. S1. 127 We thank the reviewer for pointing this out. We have corrected it in the revised manuscript. 128

Clarity and context: 129
The ideas are very easy to follow as they are clearly explained step-by-step and the flow 130 follows a logical sequence. Additionally, sufficient context have been provided to understand 131 the rationale of each experiment. 132 We thank the reviewer for her/his positive comments. 133 References: The references are appropriate. 136 We thank the reviewer for her/his positive comments. In this manuscript the authors present a detailed investigation of the how contact with binding 157 partners controls the conformation and function of β-arrestin 1. First the authors evaluate how 158 the binding of phosphorylated peptides either with or without a key phosphorylated residue (at 159 position T360) or the synthetic intrabody (Ib30) impact arrestin conformation, as measured 160 using a limited trypsin proteolysis assay. These assays suggested subtle conformational 161 differences were induced through arrestin binding as demonstrated by differences in the extent 162 and site of trypsin proteolysis. These differences were evaluated in the context of previously 163 published structural data as well as new molecular dynamics studies. The authors next 164 evaluated functional consequences of the interaction between the synthetic intrabody and 165 beta-arrestin in live cells. Here they found that Ib30 expression facilitates endosomal trafficking 166 upon V2R T360A activation, a very surprising and interesting finding. This effect of Ib30 was 167 shown to correlate with an enhanced interaction between beta-arrestin and beta-adaptin, 168 which is known to facilitate internalization. 169 We thank the reviewer immensely for her/his time to review our manuscript so thoroughly and 170 for her/his positive comments. 171 Overall, the paper is well written, the experiments are carefully performed and interpreted 172 appropriately, and the biological implications of the findings are high. Some of the arguments 173 made regarding structural changes are a bit speculative. The importance of understanding 174 (and controlling) receptor internalization mechanisms is extremely high. As such, I think the 175 manuscript could be suitable for publication provided some major and minor concerns are 176 addressed. 177 We thank the reviewer for her/his positive comments and important feedback. 178 Major comments: 179 *Receptor internalization (and signaling from internalized compartments) is increasingly being 180 recognized as an important element in determining the biological output of receptor activation.

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The system described here seems to offer a way to toggle internalization on/off. However, this 182 manuscript doesn't address whether the internalization observed for bArrestin in cells 183 expressing V2R-T360A and Ib30 also correlates with receptor internalization. The authors 184 describe already use a whole cell ELISA assay for looking at receptor levels. This assay (or 185 something related) should be used to see whether Ib30 also induces receptor internalization 186 for V2R-T360A. This would substantially improve the impact of this manuscript. 187 This is a very interesting point. Following the reviewer's suggestion, we measured the 188 endocytosis and trafficking of V2R T360A using whole cell ELISA and NanoBiT assays. While we 189 expected that receptor endocytosis would follow a similar pattern as βarr1 trafficking, we 190 surprisingly observed that V2R T360A internalizes and localizes to endosomes as efficiently as 191 V2R WT (please see Figure R2.1 below). These data are quite intriguing as they seem to 192 suggest separate trafficking of V2R and βarr1 after agonist-stimulation. We believe however 193 that this interesting lead requires substantial additional investigation as a follow up study, and 194 we Following the reviewer's suggestion, we have now measured surface and total recruitment of 202 βarr1 as well as the endosomal trafficking of βarr1 using NanoBiT assays. We have now 203 included these data as Figure 4A-D (presented below as Figure R2.2) and Figure 6 and S5 204 (presented below as Figure R2.2). The endosomal trafficking data using NanoBiT assay 205 essentially recapitulate the pattern observed using the BRET assay. conformations for dynamic proteins is tricky and predicting binding for antibody (fragments) is 210 also tricky. Can the authors provide more evidence that the modeling they performed for 211 bArr1/VwRppT360A/Fab30 is likely to be reflective of reality? As it stands now, I feel the 212 manuscript makes too strong of a conclusion from a modelling experiment that is filled with 213 uncertainty. 214 We completely understand the point made by the reviewer. However, we would like to 215 underscore that our simulation studies are based on previously determined high-resolution 216 crystal structures of βarr1-V2Rpp WT /V2Rpp T360-1 /Fab30 complexes (PDB: 4JQI and 7DFA). It is 217 important to note that these crystal structures have provided an excellent understanding of the 218 Fab30 binding interface on βarr1 and the inter-domain rotation, which guided our molecular 219 dynamics simulation studies. In fact, our previous study has revealed that T 360 A mutation in 220 V2R shifted the conformational equilibrium of βarr1 towards an inactive state characterized by 221 smaller inter-domain rotation, an observation we have recapitulated in the current study as 222 well. Moreover, another recent study based on V2Rpp-βarr1-Fab30 structure has reported that 223 the removal of the phosphopeptide resulted in βarr1 inactivation with respect to inter-domain 224 rotation angle. As the binding interface of Fab30 spans both, the N-domain and C-domain in 225 βarr1, its positive effect on inter-domain rotation, as observed in our simulation studies, is 226 highly anticipated. Finally, a recent study has measured the effect of Fab30 on βarr1 227 conformation using solution NMR methodology, and converged onto a similar conclusion in 228 terms of stabilizing effect of Fab30 on βarr1 activation as observed in our simulation study 229 (30). Therefore, we believe that our simulation data provide important insights into the 230 allosteric effect of Fab30 on βarr1 activation with respect to inter-domain rotation that can be 231 linked to βarr1 trafficking and signaling. Still however, following reviewer's feedback, we have 232 now included a brief discussion on this in the revised manuscript (page 11-12, line 271-276).

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Minor comments: 235 *For all statistical comparisons a one-way ANOVA is used to provide p-values but this test 236 requires post-hoc analysis (like Tukeys) to inform you which groups are actually statistically 237 different from each other. Could the authors specify which post-hoc tests were used? It would 238 also be helpful to specify which two groups are being compared for each p value (I think I can 239 follow but I don't want to have to assume). This is an interesting point. As the phosphopeptides are only 29 amino acid long, it is difficult 250 to follow their proteolysis patterns using SDS-PAGE, and it will require more sophisticated 251 approaches(e.g. mass spectrometry). However, we have analyzed the phosphopeptide 252 sequence in-silico, and as presented in Figure R2.3 below, the trypsin cleavage sites are 253 present only at the edges in these peptides. In other words, even if the peptides were being 254 degraded differentially by trypsin, it will not influence the proteolysis pattern of βarr1 as the 255 core segment of the phosphopeptides harbouring the phosphorylated residues would remain 256 intact. 257 258 *In Figure 1c why is there a band labeled Gly(-8) to Arg418 that doesn't appear in Figure 2c? 259 The 48kDa band is not apparent in Figure 2C as the time-point (post-trypsin addition) for this 260 experiment is 30min (in contrast with Figure 1C where the time-point is 5min), at which the 261 48kDa band is completely digested. We have now indicated the time points in the respective 262 figure legends to clarify this point. 263 *What accounts for the reduced ability of ScFv30 to immunoprecipitate beta-arrestin 264 1/V2RppT360 complex (Figure 2a)? Is the phosphopeptide used at a saturating concentration 265 for beta-arrestin here (what is the KD for this interaction?) Is it because ScFv30 has a lower 266 affinity for beta-arrestin 1/V2RppT360 complex? If this is the case could you explain it explicitly 267 in the text? 268 This is an interesting point and we have observed this pattern for both, ScFv30 and Fab30. In 269 these co-IP experiments, we used phosphopeptides at two different molar excess ratios i.e. Here, we have monitored the localization of βarr1 using confocal microscopy in different fields 281 of view and manually scored βarr1 localization from more than 500 cells for each condition.

282
These data are presented as % normalized i.e. what % of total cells display membrane 283 localization of βarr1 vs. punctate localization. Here, three independent replicates represent 284 three different transfections on different days, their confocal imaging and manual scoring of 285 βarr1 localization from pool of cells from these three experiments. Following reviewer's 286 suggestion, we have now clarified this in the revised manuscript (page 9, line 202-203 and 287 page 21-22, line 519-527 and revised figure legend of Figure 5F). 288 *The meaning of the data presented in the inset in Figure 4b is unclear. Could the authors 289 expand on the explanation of this data or plot it in a different way? 290 We regret the lack of clarity, and we have now plotted this data differently. We have essentially 291 shown the change in BRET signal (ΔBRET) for all four conditions in Figure 6C. The key point 292 in this graph is to demonstrate the effect of Ib30 on endosomal trafficking of βarr1 in terms of 293 maximal agonist-induced response i.e. change in BRET signal. We have also clarified this 294 point in the revised figure legend. 295 *It seems the level of Ib-CTL/30 is variable in Figure 4c and no conventional loading control is 296 shown. Can the authors comment on this? 297 Although we have expression normalized Ib-CTL and Ib30, there is a slight variation in their 298 relative expression levels in some experiments. But, despite a slightly lower expression level, 299 Ib30 exhibits a robust positive effect on ERK1/2 activation, which further strengthens the point. 300 We had not included conventional loading control e.g. β-actin as the total-ERK1/2 blot itself 301 serves as a loading controls. Still however, following reviewer's suggestion, we have now 302 repeated the experiment and show β-actin loading control as well (presented below as Figure  303 R2.4; and included as Figure 7A in the revised manuscript). 304 305 *Could the authors comment on whether endogenously expressed beta-arrestins play any role 306 in the observed results for cell-based assays? 307 This is an interesting point. However, in most of the cellular experiments except ERK1/2 assay, 308 we are using exogenous βarr1 that is engineered for the corresponding assay (e.g. 309 SmBiT/LgBiT-tagged in NanoBiT assay, mCherry/mYFP-tagged in confocal imaging and R-310 Luc-tagged in BRET assay). Therefore, the measured responses arise specifically from the 311 exogenous βarrs and not from endogenous βarrs. However, we cannot rule out the possibility 312 that endogenous βarrs may compete for the receptor upon agonist-stimulation but that is likely 313 to be similar across different conditions, if any. 314 *Why is the non-specific band observed in Figure 6A not seen in other blots? 315 We thank the reviewer for pointing this out. In this experiment, we have used lysate from Sf9 316 cells expressing V2R T360A while in other experiments ( Figure 7A and Figure 9E), we have used 317 HEK-293 cells. We have observed this non-specific band in anti-HA blots when using lysate 318 from Sf9 cells in other experiments as well in our laboratory. We have now clarified in the 319 revised figure legend. 320 321 322

Reviewer #3: 323
In their manuscript, the authors investigate the effect of the intrabody30 (Ib30) on the activity 324 state of arrestin bArr1 bound to the V2R. The manuscript builds on previous observations 325 published in Nat. com., that the T360A mutation at the C-terminus of the receptor leads to a 326 loss of arrestin translocation to the endosomal membrane due to a prominent shift in arrestin 327 from active to inactive. In the current manuscript, they investigate the effect of Ib30 binding to 328 bArr1 to reverse this effect. For this purpose, they use various biochemical and biophysical in 329 vitro and in silico methods. In their experimental setup the authors find that Ib30 rescues 330 agonist induced ERK 1/2 MAP kinase activation by the T360A mutation. Moreover, that Ib30 331 enhances interaction of bArr1 with b2-adaptin. To the reviewer, the experiments appear well 332 presented and convincing. 333 We thank the reviewer for her/his positive comments. 334 In the second part of the manuscript, structural explanations are provided. This portion is of 335 particular importance for the argumentation, as it establishes a reference to the structural level. 336 However, in the opinion of the reviewer, this part is not elaborated far enough and should be 337 deepened or better documented in order to make it accessible and understandable to the 338 broad readership of the journal. The MD simulations indeed seem to have the potential to 339 reproduce and substantiate the observations at a structural level. The interdomain rotation 340 angle used here is a recognized measure for classifying a state as active or inactive. In the 341 present manuscript, the authors use this very measure to support the above hypothesis. The 342 results of three simulation repeats are summarized in Fig. 5B in the form of a histogram. The 343 current presentation of the data appears to the reviewer to be inadequate because significance 344 is not apparent from the provided histograms. However, the data provided to the reviewer on 345 request during the review process allows the following assessment of the analyses presented: 346 The MD simulations indeed confirm the hypothesis, since within the limitation of non-sufficient 347 sampling, the WT arrestin tends to be in active conformation, the T to A mutation tends to be 348 in inactive conformation, and the latter mutation in complex with intrabody30 (Ib30) again 349 tends to be in active conformation. From the running averages now available to the reviewer, 350 it is clear that the arrestin does not remain in one state in each case, but rather fluctuates 351 between active and inactive, which has not previously been described in this way in the results 352 section. This fluctuation proves that the respective tendency to active or inactive, is not an 353 artifact of a single simulation, but rather and despite the limited simulation time describes an 354 actual property of the system. The reviewer suggests to integrate the histograms into the 355 illustration of the running averages, for example the present illustration turned by 90°, at the 356 end of this. This would already make clear from the figure that the undoubtedly different 357 tendencies are due to an overall limited amount of data -in this case three short simulations -358 and do not represent the actual ensemble. In doing so, the dimensions of the Y-axes should 359 also be aligned for all three systems to ensure direct comparability. Furthermore, a space 360 between the individual trajectories should convey even better to the reader that we are dealing 361 with three independent runs and not a continuous simulation. In any case, this limitation, which 362 most simulation work is subject to due to the limiting and costly calculations, should be 363 sufficiently described in the paper. 364 We very much appreciate reviewer's time and effort to carefully review our data, and provide 365 constructive feedback to strengthen our manuscript. Following reviewer's suggestion, we have 366 now integrated the histograms of the inter-domain rotation angle into the illustration of their 367 running averages (please see Figure R3.1 below). This figure is now included as Figure 8B-D 368 in the revised manuscript. 369 370

372
Regarding sampling limitations of MD simulation, we agree with the reviewer that current 373 simulation times may have limitations in obtaining a converged sampling of the conformational 374 space of βarrs. To clarify this point to the readers, we have now included a brief discussion of 375 this the revised manuscript (page 11-12, line 271-276). Moreover, to further support our 376 findings, we have now increased the sampling to an accumulated time of 10µs per system 377 (i.e. 5 runs of 2µs each), which strengthens our observations. We have revised the main text 378 (line 271-272), method section (page 25, line 597-600) and the corresponding figure legend 379 ( Figure 8B) accordingly. 380 The reviewer wonders why the structural interpretations from Figs. 2E, F and 5A based on 381 previously published crystal structures, were not supported with MD simulations. On the one 382 hand, the view on static structures does not allow optimal statistics of the contacts, i.e. it is not 383 clear whether the structural interpretations mentioned in the text, which are partly due to side 384 chain movements of single amino acids, are equally observable in a dynamic setup. Second, 385 and more importantly, the described effects of the T to A mutation on the activation or 386 deactivation of arrestin or the effect of the intrabody30 (Ib30) thus do not experience a 387 sufficient structural explanation. A more detailed study of the structures using MD, or further 388 analysis of the existing MD data, should allow the authors to describe the long-range allosteric 389 effects underlying their observations. Naturally, the MD part would thus assume an even more 390 importance in the manuscript. 391 The reviewer makes an interesting point here. The structural snapshots included in Figure 3A 392 (previously 2E-F) were primarily presented to indicate the orientation/positioning of Arg 285 and 393 Arg 188 , two trypsin cleavage sites in βarr1 resulting in 32kDa and 21kDa band, respectively, 394 based on the previously published crystal structures. Similarly, the structural snapshot in 395 Figure 8A (previously 5A) were presented to highlight the repositioning of the proximal 396 segment of V2Rpp and the interaction of T 359 /T 360 in the two crystal structures i.e. V2Rpp  bound and V2Rpp T360-1 -bound βarr1.

398
Following the reviewer's suggestion, we have now carried out additional simulations to 399 sample the conformation of Arg 285 and Arg 188 under three different conditions i.e. basal βarr1, 400 V2Rpp WT -bound βarr1 and V2Rpp T360-1 -bound βarr1 (please see Figure R3.2 below). These 401 simulations essentially suggest that the overall conformational space sampled by these two 402 residues in V2Rpp WT -bound βarr1 and V2Rpp T360-1 -bound βarr1 states are mostly similar. This 403 observation matches well with our experimental observations ( Figure 2E), where the WT and 404 mutant peptides induce identical patterns of proteolysis with respect to the 32kDa and 21kDa 405 bands. We thank the reviewer for making this excellent suggestion that has helped us 406 rationalize the experimental data better. We have now included these simulation data as 407 Figure 3B in the revised manuscript. 408 In addition, we have also expanded the corresponding results sections to include more 409 mechanistic insight by linking the previous observations with those uncovered in the current 410 study (page 6-7, line 144-151). 411 412

414
Finally, the methodology section appears to the reviewer to be in urgent need of revision, as 415 essential information cannot be found here. It was neither helpful for the review, nor does it 416 facilitate the readability of the manuscript, if essential details are not found in the manuscript 417 but in another publication, which is merely referred to. The method used to measure the 418 interdomain rotation in the trajectories is thus not transparent, mainly because the paper they 419 cite does not explicitly state information such as: which axis they use to measure the rotation 420 angle or which reference points (selected residues/atoms or center of mass/center of mass of 421 the selected residues?). 422

423
We thank the reviewer for pointing this out, and we have now included the corresponding 424 information in the method section of the revised manuscript (page 25, line 602-609).

426
Likewise, the information on the PDB-entries, which provided the structure coordinates for the 427 respective simulations, is missing. What changes were made to the structures (mutations, 428 etc.)? Thus, it was laborious for the reviewer to gather this information from other parts of the 429 manuscript. The reader should be spared this effort.

431
We thank the reviewer for pointing this out, and we have now included the corresponding 432 information in the method section of the revised manuscript (page 25, line 604-606).

434
A final question arises from the data in the methodology section. The authors used the NVT 435 ensemble for the production runs, which is rather unusual. Why were this ensemble chosen? 436 437 The choice of the NVT ensemble was primarily motivated by the simulation package we used 438 ACEMD [PMID: 26609855]). As ACEMD only enables the usage of the Berendsen barostat, it 439 is a good choice for equilibration but not production runs, ACEMD users switch to an NVT 440 ensemble for production runs. Although an NPT ensemble is intuitively more correct for 441 production runs of biological systems, ACEMD developers state that "With the system sizes 442 which are achievable nowadays, it is not necessary to have a pressure control in the 443 production runs …. (for large number of atoms all ensembles are equivalent, statistically)".

445
In summary, this manuscript describes work with potential interest for the broad readership of 446 the journal. The experimental part of the work seems convincingly elaborated to the reviewer. 447 The structural level is of great importance for the verification of the biochemical analyses and 448 substantial for the understanding of the effects described here. There are still deficiencies 449 here, but these could be solved within the interdisciplinary orientation of the authors. It is 450 important for the reader to understand not only that the T to A mutation has an effect on the 451 dynamic equilibrium of inactive to active arrestin states, but also how this effect comes about 452 structurally and how it can be reversed by binding of intrabody30 (Ib30). At this point, further 453 analysis seems necessary. Finally, the MD part should be described and documented in more 454 detail.

456
We thank the reviewer for his/her positive comments and constructive suggestions. As 457 mentioned above against specific comments/suggestions, we have now addressed all the 458 points raised by the reviewer in the revised manuscript.

460
The working group around Jana Selent has created a useful basis for sharing MD data with 461 the implementation of GPCRMD. I would expect her to use this to share the trajectories 462 analyzed here with the reviewers adequately and subsequently make them available to the 463 community in the spirit of the FAIR principles.

465
We most certainly support data sharing and transparency, and following reviewer's excellent 466 suggestion, we have submitted our simulation data to the GPCRMD portal 467 ( in these provided gels any conclusion on kinetic effects for the digest will depend on the 502 amount of arrestin added in the individual sample and this is not quantified, at least I did not 503 see this. Hence, this part of discussion is rather weak if not over interpreted. 504 We understand reviewer's point but there appears to be a slight misunderstanding here. In 505 these experiments, we prepare βarr1+V2Rpp samples in parallel, and took out an aliquot 506 before adding trypsin. Subsequently, we add trypsin to the samples followed by incubation at 507 37°C for 5min, and take out another volume-normalized aliquot (i.e. accounting for slight 508 volume change with trypsin addition). We did the same for all samples (i.e. apo-condition and 509 phosphopeptide-bound) in parallel, and then run trypsin-digested samples for all conditions 510 side-by-side. Still however, we have now repeated the experiment under apo and V2Rpp WT 511 conditions, and ran samples before and after proteolysis for each condition side-by-side to 512 demonstrate equal starting material in each condition ( Figure R4.1). As evident from the band 513 intensity, the starting amount of protein is comparable for each time-point. 514 3) In figure 4 B the V2R wt conditions without and with Ib30 seem to be only shifted in parallel 516 upwards, please take this into consideration in the discussion. The effect might not be as big 517 as described if the two curves would start from the same basal level…. 518 We thank the reviewer for pointing this out, which allows us to further clarify this in the main 519 text. In fact, we did not focus here on the V2R WT , which as the reviewer correctly points out, 520 does not change significantly between Ib-CTL and Ib30 conditions, if the parallel shift is taken 521 into account. Instead, we underscore the effect of Ib30 on V2R T360A mutant; where the basal 522 between Ib-CTL and Ib30 conditions are identical but the maximal responses are dramatically 523 different (compare the line with blue circle to blue square symbols). We have now elaborated 524 on this further in the main text (page 9, line 212-215) and also plotted the ΔBRET data 525 differently to make this point better ( Figure 6C in the revised manuscript). 526 4) Figure 5 is of concern as well. The modeling is nice but the major effect of Ib30 stabilizing 527 the active conformation of arrestin was shown before using Fab30, the parental antibody of 528 Ib30. This was shown by stabilizing NMR signals of arrestin (cited as Ref. 26). Hence, again 529 the novelty is limited since this result was to be expected, although it is great to see this 530 stabilization in living cells and not only NMR. 531 We thank the reviewer for appreciating our modelling and cellular data. Regarding the aspect 532 of novelty, we would like to underscore that in the previous study, we had reported that the 533 inter-domain rotation induced in βarr1 upon V2R T360A mutation is significantly reduced 534 compared to that by the V2R WT . We had not explored, or commented on, the influence of Fab30 535 (or, ScFv30) on the inter-domain-rotation, an aspect that we probe and describe in the current 536 manuscript for the first time. The NMR data that the reviewer appropriately refers to, 537 demonstrates the positive effect of Fab30 on promoting a fully-engaged-like conformation of 538 βarr1 upon interaction with a chimeric β2-adrenergic receptor (referred to as β2V2R). On the 539 other hand, the modelling data included in the current study uncovered a positive allosteric 540 effect of Ib30 on inter-domain rotation in βarr1 for a single phospho-site mutant of the V2R. 541 5) In figure 6 C the V2R T360A conditions without and with Ib30 seem to be only shifted in 542 parallel upwards, this needs to be taken into account. If the curves would start at the same 543 basal level they would be identical and Ib30 had no effect for the T360A mutant… 544 We agree with the reviewer but there appears to be another misunderstanding between us. In 545 fact, our co-IP data (presented in Figure 9A) suggest that ScFv30 enhances the basal 546 interaction between βarr1 and β2-adaptin. This is also reflected in cellular data using BRET 547 ( Figure 9C) where we observe an enhanced basal signal for both, V2R WT and V2R T360A in 548 presence of Ib30 (compared to Ib-CTL). Based on this, we designed the Ib30 titration 549 experiment presented in Figure 9E, where we observed a saturable response in βarr1-β2-550 adaptin interaction with increasing Ib30 expression level. Taken together, these data establish 551 the positive effect of ScFv30/Ib30on βarr1-β2-adaptin interaction, and we have now clarified 552 this in the revised manuscript (page 12-13, line 296-300). 553

Minor points: 554
For this manuscript it would also be helpful to clearly state in the manuscript the differences 555 or similarities between T360A-1 and -2 and when they can be summed up to T360A to follow 556 the authors' throughout the manuscript. 557 We thank the reviewer for mentioning this. We refer to the phospho-peptides in Figure 1 and 558 2 as V2Rpp T360-1 and V2Rpp T360-2 , and we refer to the receptor mutant as V2R T360A . We have 559 now clarified this in the revised manuscript (page 4, line 90-94). 560 Please make sure that all needed information about statistical analysis is clearly and 561 consistently stated in all figure legends etc. throughout the manuscript. 562 We have carefully revised the figure legends to include all the information related to statistical 563 analysis. 564 Line 27, 63/64 vasopressin 2 receptor or vasopressin type 2 receptor 565 We have corrected this in the revised manuscript. 566 Line 76 previously described intrabody (Ib30)-based sensor (hyphen). 567 We have corrected this in the revised manuscript. 568 Line 127 additional information to the CONTACT/ACT program would be nice in order to make 569 it more understandable to readers who are not so familiar. 570 We have now included additional details about this in the revised manuscript (page 6, line 571 140-143). 572 Line 211 Please elaborate on the ear-domain of β2-adaptin for the less conversed readers. 573 We have corrected this in the revised manuscript (page 12, line 281-285). 574 We have now included this information in revised Figure 1B. 577 -either reduce the shown trypsin digest data in D (because it seems like the data are shown 578 twice for the different trypsin:βarr1 ratios) or make clear why it is important to show both/ point 579 out what is the important difference/message to take from it as a reader. 580 We have included the limited proteolysis data at two different trypsin:βarr1 ratio in Figure 1D  581 in order to better highlight the differences between V2Rpp and V2Rpp T360-1/2 . For example, the 582 difference in the intensity of 48kDa and 47kDa bands are visualized better at 1:50 ratio while 583 the difference in the 32kDa and 21kda bands are visualized better at 1:25 ratio. We have now 584 explained this in the revised manuscript (page 5, line 102-104). 585 -of note: significance level is set to * p < 0.1 instead of 0.05… (which is not the case in Fig.2  586 for example) 587 We have now corrected this in the revised manuscript. 588

Figure 2 589
-it would be nice to have the probes in 2C consistently with 1C to make it easier for the reader 590 We have now corrected this in the revised manuscript. 591 -please indicate whether T360-1 or -2 are shown in Fig.2D or whether it is a representation 592 for both, the Fig.2 focusses on T360-1 in E and F, is this also the case in D? 593 Figure 2E reflects the pattern common for both, V2Rpp T360-1 and V2Rpp T360-2 . We have now 594 corrected this in the revised figure and the figure legend. 595 -in figure 2c please also indicate if the big band above 29kDa is the ScFc30 or something 596 else 597 This band is indeed ScFv30, and we have now indicated it in the revised Figure 2E. 598 in the supplements where they do not co-express the receptor of interest to clearly show that 601 the observed effect is dependent on the presence of the receptor. 602 In these experiments, we are measuring the response (change in luminescence signal in 603 Figure 5B and localization of βarr1 in Figure 5C and E) upon stimulation of cells with agonist. 604 Therefore, the observed effects should clearly be based on receptor activation. Still however, 605 following reviewer's suggestion, we have now repeated these experiments without receptor 606 expression. As presented below in Figure R4.2, we do not observe the corresponding 607 responses in cells lacking the receptor. 608 609 Figure 4 610 -in Figure 4D, the statistical comparison of V2R-T360A + Ib30 to the WT receptor would be 611 also interesting to add statistical relevance to the statement "it robustly enhanced the level of 612 phosphorylated ERK1/2 upon agonist-stimulation, nearly to that of the V2RWT" (line 177/178) 613 Following reviewer's suggestion, we have now compared Ib30 conditions for V2R WT and 614 V2R T360A and included this in the revised Figure 7D. 615  Fig.6 to support the statement "There was robust 617 interaction between βarr1 and β2-adaptin for the V2RWT upon agonist-stimulation in the 618 presence of control intrabody, while the response was significantly lower for V2R T360A" (line 619 221-223) and also in lines 224-227. 620 Following reviewer's suggestion, we have now compared Ib-CTL conditions for V2R WT and 621 V2R T360A and included this in the revised Figure 9B. We have also compared the basal and 622 maximal response for βarr1-β2-adaptin interaction in the BRET assay ( Figure 9C) and present 623 this analysis in Figure 9D. 624 We have now included the quantification of the 48kDa band in the revised Figure S1 and 628 indicated the time-point in the figure legend. 629 -of note: significance level is set to * p < 0.1 instead of 0.05… (which is not the case in Fig.S2  630 for example) 631 We have now corrected this in the revised figure legend. 632 We have now corrected this in the revised figure legend. 638 Figure S4  639 -was the significance level set to ns p> 0.1 or > 0.05 here? 640 The significance level for ns was set to p>0.05, and we have now included this in the revised 641 figure legend. 642

Reviewer #1 2
Almost all the recommended improvements have been done in the new version of the 3 manuscript, except one. It seems that the authors forgot to address the concern about the 4 analytic approach (see lines 62 to 67 in the rebuttal). Consequently, I recommend this 5 manuscript to be published conditionally to addressing this concern. 6 We thank the reviewer for her/his positive comments, and we apologize for missing the point 7 about statistical test. In fact, we have compared One Way ANOVA and t-test on these data 8 sets, and the overall significance including p values does not change significantly. As we are 9 comparing more than two groups measured concurrently in these experiments, we believe 10 that using One Way ANOVA for statistical analysis is appropriate. 11

Reviewer #2 12
The authors have done a thorough job of responding to my comments. I think the revised 13 manuscript is well-suited for publication. There is one author response on which I request 14 clarification. I can understand the need not to include every interesting follow up finding in this 15 manuscript, but I am concerned the way the revised manuscript is written leads readers in the 16 opposite direction from the findings the authors provided to reviewers. The authors provided 17 data for reviewers showing that V2RT360A DOES NOT follow a pattern of agonist induced 18 endocytosis similar to that of bArr1. If the authors are going to speculate on this, they should 19 at least speculate about what they already know to be supported by data. If the authors are 20 not going to include these new findings, they should update this section of the text to lead 21 readers in the direction supported by their preliminary findings. 22 We thank the reviewer for raising this insightful point, and following her/his suggestion, we 23 have modified the corresponding text accordingly (line 330-337, page 14). 24 Original text: 25 For example, in case of wild-type V2R, agonist-stimulation promotes co-localization of the 26 receptor, βarr1 and Ib30 in endosomal vesicles 13 ; however, this remains to be determined for 27 the V2R T360A mutant in absence and presence of Ib30. If agonist-induced endocytosis of 28 V2R T360A also displays a pattern similar to that of βarr1 trafficking, it will be important to 29 understand whether Ib30 rescues receptor endocytosis as well. This may also help clarify the 30 underlying mechanism for the lack of cAMP potentiation in case of V2R T360A despite enhanced 31 endosomal trafficking of βarr1 in the presence of Ib30. 32

Revised text: 33
For example, in case of wild-type V2R, agonist-stimulation promotes co-localization of the 34 receptor, βarr1 and Ib30 in endosomal vesicles 13 ; however, it is plausible that V2R T360A 35 dissociates from βarr1 at the plasma membrane due to relatively lower affinity. This may lead 36 to trafficking of βarr1, presumably stabilized in an active conformation by Ib30, to endosomal 37 vesicles even in the absence of the receptor. Further investigation along these lines in future 38 studies may also help clarify the underlying mechanism for the lack of cAMP potentiation in 39 case of V2R T360A despite enhanced endosomal trafficking of βarr1 in the presence of Ib30. 40 Reviewer #3 41